Heterotrophic cultivation of Chlorella sp. HS2 for lipid production and its transcriptome analysis

Abstract

Heterotrophic cultivation has notable benefits, including high cell density and the ability to produce biomass in a consistent and predictable amount and the relatively low risk of contamination. Heterotrophic cultivation of Chlorella sp. HS2 was optimized in this study. First, various sources of carbon and nitrogen were selected in the photobiobox. Next, Plackett-Burman Design (PBD) was used to check the effect of each nutrient on cell growth. And the concentration of the major nutrients (glucose, nitrate, and phosphate) was optimized through the Response Surface Methodology (RSM) using the Central Composite Design (CCD). The model verification results through flask cultivation showed that the modified BG11 medium improved the total cell production by 3-fold from 5.85 to 18.13 g $L^{-1}$ compared to the normal BG11 medium. Expanding the incubation scale to a 5-L fermenter greatly improved biomass concentration of 35.3 ± 0.6 g $L^{-1}$ due to more efficient cultivation. Compared with other Chlorella spp., the biomass concentration was significantly higher, but the lipid content was low. To overcome these problems, I tried to solve them through fed-batch cultivation. The BG11 medium was also used to do fed-batch cultivation by starting incubation and adding the optimized BG11 medium. As a result, the biomass concentration was increased from 32 g $L^{-1}$ to 50 g $L^{-1}$. In the case of lipid content, it also increased sharply from 26% to 40%. But there were some weak parts here, too. In addition to glucose, there was a buildup of other nutrients. So I tried again by adding nutrients only the required amount of biomass to the target biomass concentration. And the dissolved oxygen was also found to have affected cell growth by falling from days 2-3 to zero. In order to solve this problem, I want to gradually increase RPM. As a result, biomass grew beyond 50 g $L^{-1}$ in just 5 days, and existing biomass production doubled. Dissolved oxygen was kept above 20%. Glucose, as it had been cultured earlier, maintained a good 20-40 g $L^{-1}$ and nitrate remained after 4 days. On the 8 day, when lipid production was the highest, lipid production was 2.29 g $L^{-1}$$d^{-1}$, an improvement from 1.99 g $L^{-1}$$d^{-1}$, the lipid productivity of the fed-batch cultivation initially conducted. However, lipid content decreased by about 7.6% points from 39.8% to 32.2%. In order to increase lipid production, nitrate was given in half to try the fed-batch cultivation with nitrogen depletion stress again. As a result, nitrate was depleted in 3 days and lipid production edged up to 2.31 g $L^{-1}$$d^{-1}$ on the 8 days when lipid production was the highest. Here, biomass grew rapidly to 47 g $L^{-1}$ in just 3 days. The transcriptome analysis was attempted to further interpret the difference in lipid accumulation process seen in the fed-batch of the nitrogen replete and depleted conditions. As a result, we successfully increased total lipid productivity in the Fed-batch experiment. Then, we predicted and identified phenotype of N-limited cultivation by confirming expression level of N-limited mRNA. We tried transcriptome analysis to explain the special regulatory mechanism about N-limited cultivation. Then, quantified gene expression between N-sufficient and N-limited cultivation was compared, and gene ontology was conducted. Based on these data, we predicted specific genes that could be regulated by Chlorella sp. HS2 under specific culture conditions. These results suggest new possibilities of upgraded cultivation process engineering of Chlorella sp. HS2 for the production of valuable bio-product and biofuels.